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1.
Cytometry B Clin Cytom ; 106(1): 11-24, 2024 01.
Article in English | MEDLINE | ID: mdl-38345160

ABSTRACT

The 5-azacytidine (AZA) and decitabine (DEC) are noncytotoxic, differentiation-inducing therapies approved for treatment of myelodysplastic syndrome, acute myeloid leukemias (AML), and under evaluation as maintenance therapy for AML postallogeneic hematopoietic stem cell transplant and to treat hemoglobinapathies. Malignant cell cytoreduction is thought to occur by S-phase specific depletion of the key epigenetic regulator, DNA methyltransferase 1 (DNMT1) that, in the case of cancers, thereby releases terminal-differentiation programs. DNMT1-targeting can also elevate expression of immune function genes (HLA-DR, MICA, MICB) to stimulate graft versus leukemia effects. In vivo, there is a large inter-individual variability in DEC and 5-AZA activity because of pharmacogenetic factors, and an assay to quantify the molecular pharmacodynamic effect of DNMT1-depletion is a logical step toward individualized or personalized therapy. We developed and analytically validated a flow cytometric assay for DNMT1 epitope levels in blood and bone marrow cell subpopulations defined by immunophenotype and cell cycle state. Wild type (WT) and DNMT1 knock out (DKO) HC116 cells were used to select and optimize a highly specific DNMT1 monoclonal antibody. Methodologic validation of the assay consisted of cytometry and matching immunoblots of HC116-WT and -DKO cells and peripheral blood mononuclear cells; flow cytometry of H116-WT treated with DEC, and patient samples before and after treatment with 5-AZA. Analysis of patient samples demonstrated assay reproducibility, variation in patient DNMT1 levels prior to treatment, and DNMT1 depletion posttherapy. A flow-cytometry assay has been developed that in the research setting of clinical trials can inform studies of DEC or 5-AZA treatment to achieve targeted molecular pharmacodynamic effects and better understand treatment-resistance/failure.


Subject(s)
Leukemia, Myeloid, Acute , Leukocytes, Mononuclear , Humans , Decitabine/pharmacology , Decitabine/therapeutic use , Flow Cytometry , Reproducibility of Results , Azacitidine/pharmacology , Azacitidine/therapeutic use , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Biomarkers
2.
Semin Hematol ; 58(1): 45-55, 2021 01.
Article in English | MEDLINE | ID: mdl-33509443

ABSTRACT

The DNA methyltransferase inhibitor azacytidine (aza) may reactivate pathways associated with plasma cell differentiation, cell cycle control, apoptosis, and immune recognition and thereby restore sensitivity to lenalidomide (len) and dexamethasone (dex) in relapsed and/or refractory multiple myeloma (RRMM). We aimed to develop an aza regimen that reaches epigenetically active levels 8 times in 28 days with less bone marrow toxicity than the myeloid malignancy standard of 7 consecutive doses to enable safe combination with len. Aza was escalated from 30 mg/m2 once a week up to a predefined maximum of 50 mg/m2 twice a week in combination with GFR-adjusted len (≥ 60 mL/min: 25 mg, 3059 mL/min: 10 mg) day 1 to 21 every 28 days and dex 40 mg once a week followed by a limited expansion study to a total N of 23 at the highest tolerated dose. Fifty-one patients (pts) with RRMM were screened, 42 were treated and 41 were evaluable for response based on at least 1 response assessment or progression after treatment start. The median number of prior lines of therapy was 5 (1-11) and 81% (34) were refractory to len and/or pomalidomide (pom). Two DLTs occurred in different cohorts, 1 neutropenic fever in 1/6 pts on the aza 40 mg/m2 twice a week GFR ≥ 60 mL/min cohort and 1 GGT elevation in 1/6 pts on the aza 50 mg/m2 GFR 30-59 mL/min cohort. An MTD was not reached and aza 50 mg/m2 SC twice a week was chosen for the expansion study. At least possibly related Grade 3/4 AEs occurred in 28 pts (67%) with the following in > 1 pt: neutropenia (N = 16, 38%), anemia (N = 6, 14%), lymphopenia (N = 5, 12%), thrombocytopenia (N = 4, 10%), leukopenia (N = 4, 10%), febrile neutropenia (N = 4, 10%), fatigue (N = 3, 7%), fever (N = 2, 5%), and infection (N = 2, 5%). At a median follow up time for alive pts of 60.2 months (range: 36.1-82.5 months), the overall response rate (≥ partial response) and clinical benefit response rate (≥ minor response) was 22 and 32%, respectively, with 4 very good partial responses (10%), 5 partial responses (12%), and 4 minor responses (10%). The median PFS was 3.1 months (95% confidence interval [CI]: 2.1-5.1 months), median TTP 2.7 months (95% CI: 2.1-7.5 months), and median OS 18.6 months (95% CI: 12.9-33.0 months). Achieving at least minor response and reaching TTP > 6 months was associated with approximately 35% lower median plasma levels of the enzyme that inactivates aza, plasma cytidine deaminase (CDA, P< .0001). Two of the len refractory pts achieved longer disease control than with any prior regimen and 1 responded immediately after progression on len, bortezomib, and prednisone. Analyses of the methylation state of over 480,000 CpG sites in purified myeloma cells at screening were possible in 11 pts and on day 28 in 8 of them. As in other studies, the majority of differentially methylated CpGs compared to normal plasma cells were hypomethylated in myeloma. Treatment decreased the number of CpGs that were differentially methylated in normal plasma cells by > 0.5% in 6 and by > 5% in 3 of the 8 pts, most pronounced in 2 pts with clinically convincing aza contribution who achieved a reduction in overall differentially methylated CpGs by 23 and 68%, respectively, associated with increased expression of immunoglobulin genes. The study demonstrated tolerability of twice a week SC aza at 50 mg/m2 with len and dex in RRMM and suggested aza may help overcome the len/pom refractory state, possibly by activating differentiation pathways. Relatively low response rates and association of clinical benefit with low plasma levels of the aza inactivating enzyme CDA suggest the aza regimen will need to be optimized further and pt selection may be required to maximize benefit.


Subject(s)
Multiple Myeloma , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Azacitidine/adverse effects , DNA Methylation , Dexamethasone/adverse effects , Humans , Lenalidomide/pharmacology , Lenalidomide/therapeutic use , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , Treatment Outcome
3.
Br J Haematol ; 189(2): 318-322, 2020 04.
Article in English | MEDLINE | ID: mdl-31958160

ABSTRACT

T-cell large granular lymphocytic leukaemia (T-LGLL) is a chronic clonal lymphoproliferative disorder of cytotoxic T lymphocytes which commonly occurs in older patients and is often associated with autoimmune diseases. Among 246 patients with T-LGLL seen at our institution over the last 10 years, we encountered 15 cases following solid organ or haematopoietic stem cell transplantation. Here, we studied the clinical characterization of these cases and compared them to de novo T-LGLL. This experience represented a clear picture of the intricate nature of the disease manifestation and the complexities of several immune mechanisms triggering the clonal expansion.


Subject(s)
Hematopoietic Stem Cell Transplantation/adverse effects , Leukemia, Large Granular Lymphocytic/etiology , Transplantation Conditioning/adverse effects , Adolescent , Adult , Female , Humans , Leukemia, Large Granular Lymphocytic/pathology , Male , Middle Aged
4.
Br J Haematol ; 188(6): 924-929, 2020 03.
Article in English | MEDLINE | ID: mdl-31736067

ABSTRACT

The nucleoside analogue decitabine can deplete the epigenetic regulator DNA methyltransferase 1 (DNMT1), an effect that occurs, and is saturated at, low concentrations/doses. A reason to pursue this molecular-targeted effect instead of the DNA damage/cytotoxicity produced with high concentrations/doses, is that non-cytotoxic DNMT1-depletion can cytoreduce even p53-null myeloid malignancies while sparing normal haematopoiesis. We thus identified minimum doses of decitabine (0·1-0·2 mg/kg) that deplete DNMT1 without off-target anti-metabolite effects/cytotoxicity, and then administered these well-tolerated doses frequently 1-2X/week to increase S-phase dependent DNMT1-depletion, and used a Myeloid Malignancy Registry to evaluate long-term outcomes in 69 patients treated this way. Consistent with the scientific rationale, treatment was well-tolerated and durable responses were produced (~40%) in genetically heterogeneous disease and the very elderly.


Subject(s)
Antimetabolites, Antineoplastic/therapeutic use , Decitabine/therapeutic use , Myeloproliferative Disorders/drug therapy , Aged , Aged, 80 and over , Antimetabolites, Antineoplastic/pharmacology , Decitabine/pharmacology , Female , Humans , Male
5.
Sci Rep ; 9(1): 4558, 2019 03 14.
Article in English | MEDLINE | ID: mdl-30872721

ABSTRACT

The DNA hypomethylating agents decitabine and 5-azacytidine are the only two drugs approved for treatment of all subtypes of the myeloid malignancy myelodysplastic syndromes (MDS). The key to drug activity is incorporation into target cell DNA, however, a practical method to measure this incorporation is un-available. Here, we report a sensitive and specific LC-MS/MS method to simultaneously measure decitabine incorporation and DNA hypomethylation. A stable heavy isotope of 2'-deoxycytidine was used as an internal standard and one-step multi-enzyme digestion was used to release the DNA bound drug. Enzyme-released decitabine along with other mononucleosides were separated by a reverse-phase C18 column and quantified by mass spectrometry using multiple-reaction-monitoring (MRM) mode, with a lower limit of quantitation at 1.00 nM. In vitro studies demonstrated dosage and time-dependent incorporation of decitabine into myeloid leukemia cell DNA that correlated with extent of DNA hypomethylation. When applied to clinical samples serially collected from MDS patients treated with decitabine, the method again demonstrated correlation between decitabine DNA-incorporation and DNA hypomethylation. This novel assay to measure the intended molecular pharmacodynamic effect of decitabine therapy can therefore potentially provide insights into mechanisms underlying sensitivity versus resistance to therapy.


Subject(s)
DNA, Neoplasm/metabolism , Decitabine/metabolism , Endonucleases/metabolism , Exonucleases/metabolism , Leukemia, Myeloid, Acute/metabolism , Myelodysplastic Syndromes/metabolism , Animals , Antimetabolites, Antineoplastic/administration & dosage , Antimetabolites, Antineoplastic/metabolism , Apoptosis , Cell Proliferation , Chromatography, Liquid , DNA Methylation , Decitabine/administration & dosage , Humans , In Vitro Techniques , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/pathology , Mice , Myelodysplastic Syndromes/drug therapy , Myelodysplastic Syndromes/pathology , Tandem Mass Spectrometry , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
6.
Br J Haematol ; 185(5): 935-939, 2019 06.
Article in English | MEDLINE | ID: mdl-30891747

ABSTRACT

Compound heterozygous germline mutations in CTC1 gene have been found in patients with atypical dyskeratosis congenita (DC), whereas heterozygous carriers are unaffected. Through screening of a large cohort of adult patients with acquired bone marrow failure syndromes, in addition to a DC case, we have also found extremely rare or novel heterozygous deleterious germline variants of CTC1 in patients with aplastic anaemia (AA; n = 5), paroxysmal nocturnal haemoglobinuria (PNH; n = 3) and myelodysplastic syndrome (MDS; n = 2). A compound heterozygous case of AA showed clonal evolution. Our results suggest that some of the inherited CTC1 variants may represent predisposition factors for acquired bone marrow failure.


Subject(s)
Bone Marrow Failure Disorders/genetics , Germ-Line Mutation , Telomere-Binding Proteins/genetics , Telomere/genetics , Adult , Aged , Aged, 80 and over , Bone Marrow Failure Disorders/metabolism , Bone Marrow Failure Disorders/pathology , Female , Humans , Male , Middle Aged , Telomere/metabolism , Telomere/pathology , Telomere-Binding Proteins/metabolism
7.
J Clin Invest ; 128(10): 4260-4279, 2018 10 01.
Article in English | MEDLINE | ID: mdl-30015632

ABSTRACT

Nucleophosmin (NPM1) is among the most frequently mutated genes in acute myeloid leukemia (AML). It is not known, however, how the resulting oncoprotein mutant NPM1 is leukemogenic. To reveal the cellular machinery in which NPM1 participates in myeloid cells, we analyzed the endogenous NPM1 protein interactome by mass spectrometry and discovered abundant amounts of the master transcription factor driver of monocyte lineage differentiation PU.1 (also known as SPI1). Mutant NPM1, which aberrantly accumulates in cytoplasm, dislocated PU.1 into cytoplasm with it. CEBPA and RUNX1, the master transcription factors that collaborate with PU.1 to activate granulomonocytic lineage fates, remained nuclear; but without PU.1, their coregulator interactions were toggled from coactivators to corepressors, repressing instead of activating more than 500 granulocyte and monocyte terminal differentiation genes. An inhibitor of nuclear export, selinexor, by locking mutant NPM1/PU.1 in the nucleus, activated terminal monocytic fates. Direct depletion of the corepressor DNA methyltransferase 1 (DNMT1) from the CEBPA/RUNX1 protein interactome using the clinical drug decitabine activated terminal granulocytic fates. Together, these noncytotoxic treatments extended survival by more than 160 days versus vehicle in a patient-derived xenotransplant model of NPM1/FLT3-mutated AML. In sum, mutant NPM1 represses monocyte and granulocyte terminal differentiation by disrupting PU.1/CEBPA/RUNX1 collaboration, a transforming action that can be reversed by pharmacodynamically directed dosing of clinical small molecules.


Subject(s)
Granulocytes/metabolism , Leukemia, Myeloid, Acute/metabolism , Monocytes/metabolism , Mutation , Neoplasm Proteins/metabolism , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Animals , Granulocytes/pathology , Heterografts , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Mice , Monocytes/pathology , Neoplasm Proteins/genetics , Neoplasm Transplantation , Nuclear Proteins/genetics , Nucleophosmin , THP-1 Cells , Transcription Factors/genetics
8.
Mycoses ; 61(5): 321-325, 2018 May.
Article in English | MEDLINE | ID: mdl-29314351

ABSTRACT

Most superficial mycotic infections of human skin are due to dermatophytes. Children are frequently affected due to different predisposing factors, particularly overcrowding in classrooms. This study aimed to estimate the prevalence of dermatophytes infections and their related risk factors among school children in Menoufia Governorate, Egypt. Six public primary and preparatory schools were randomly selected and their pupils (n = 3464) were asked to complete a predesigned questionnaire covering both personal data and suspected risk factors for superficial dermatophyte infections. The children were also examined for dermatological diseases. Any suspected lesions were biopsied for mycological examination. The prevalence of clinically suspected dermatophytes infections was 1.41%, whereas the prevalence of culture confirmed cases was 0.98%. The most common clinical type was tinea capitis with a prevalence of 1.01%. Microsporum canis was the only isolated organism from the suspicious lesions with a 69.4% positivity rate. A higher prevalence was observed among boys, low socio-economic pupils and those with a family history of dermatophyte infections. Pet contact and sharing towels and caps among pupils were significant risk factors. Dermatophyte infection is still prevalent among basic school pupils. Fortunately, it is related to preventable risk factors. We recommend regular screening and use of educational health programmes for kids to control it.


Subject(s)
Arthrodermataceae/isolation & purification , Dermatomycoses/epidemiology , Students , Tinea Capitis/epidemiology , Adolescent , Animals , Arthrodermataceae/classification , Biopsy , Child , Cohort Studies , Crowding , Dermatomycoses/microbiology , Dermatomycoses/prevention & control , Egypt/epidemiology , Female , Humans , Male , Microsporum/isolation & purification , Pets , Prevalence , Risk Factors , Schools , Skin/pathology , Surveys and Questionnaires , Tinea Capitis/microbiology , Trichophyton/isolation & purification
10.
Korean J Pain ; 30(2): 134-141, 2017 Apr.
Article in English | MEDLINE | ID: mdl-28416997

ABSTRACT

BACKGROUND: Postoperative pain is a common, distressing symptom following arthroscopic knee surgery. The aim of this study was to compare the potential analgesic effect of dexmedetomidine after intrathecal versus intra-articular administration following arthroscopic knee surgery. METHODS: Ninety patients undergoing unilateral elective arthroscopic knee surgery were randomly assigned into three groups in a double-blind placebo controlled study. The intrathecal dexmedetomidine group (IT) received an intrathecal block with intrathecal dexmedetomidine, the intra-articular group (IA) received an intrathecal block and intra-articular dexmedetomidine, and the control group received an intrathecal block and intra-articular saline. The primary outcome of our study was postoperative pain as assessed by the visual analogue scale of pain (VAS). Secondary outcomes included the effect of dexmedetomidine on total postoperative analgesic use and time to the first analgesic request, hemodynamics, sedation, postoperative nausea and vomiting, patient satisfaction, and postoperative C-reactive protein (CRP) levels. RESULTS: Dexmedetomidine administration decreased pain scores for 4 h in both the intrathecal and intra-articular groups, compared to only 2 h in the control patient group. Furthermore, there was a significant reduction in pain scores for 6 h in the intra-articular group. The time to the first postoperative analgesia request was longer in the intra-articular group compared to the intrathecal and control groups. The total meperidine requirement was significantly lower in the intra-articular and intrathecal groups than in the control group. CONCLUSIONS: Both intrathecal and intra-articular dexmedetomidine enhanced postoperative analgesia after arthroscopic knee surgery. Less total meperidine was required with intra-articular administration to extend postoperative analgesia to 6 h with hemodynamic stability.

11.
Oncotarget ; 8(4): 6483-6495, 2017 Jan 24.
Article in English | MEDLINE | ID: mdl-28031539

ABSTRACT

BACKGROUND: Deletion of chromosome 5q (del(5q)) is the most common karyotypic abnormality in myeloid neoplasms. MATERIALS AND METHODS: To define the pathogenic molecular features associated with del(5q), next-generation sequencing was applied to 133 patients with myeloid neoplasms (MDS; N = 69, MDS/MPN; N = 5, sAML; N = 29, pAML; N = 30) with del(5q) as a sole abnormally or a part of complex karyotype and results were compared to molecular features of patients diploid for chr5. FINDINGS: A number of 5q genes with haploinsufficient expression and/or recurrent somatic mutations were identified; for these genes, CSNK1A1 and G3BP1 within the commonly deleted 5q region and DDX41 within a commonly retained region were most commonly affected by somatic mutations. These genes showed consistent haploinsufficiency in deleted cases; low expression/mutations of G3BP1 or DDX41 were associated with poor survival, likely due to decreased cellular function. The most common mutations on other chromosomes in patients with del(5q) included TP53, and mutations of FLT3 (ITD or TKD), NPM1 or TET2 and were mutually exclusive. Serial sequencing allowed for definition of clonal architecture and dynamics, in patients with exome sequencing allelic imbalance for informative SNPs facilitated simultaneous approximation of clonal size of del(5q) and clonal burden for somatic mutations. INTERPRETATION: Our results illuminate the spectrum of molecular defects characteristic of del(5q), their clinical impact and succession of stepwise evolution.


Subject(s)
Anemia, Macrocytic/genetics , Biomarkers, Tumor/genetics , Myelodysplastic Syndromes/genetics , Myeloproliferative Disorders/genetics , Anemia, Macrocytic/mortality , Anemia, Macrocytic/pathology , Case-Control Studies , Chromosome Deletion , Chromosomes, Human, Pair 5/genetics , Diploidy , Genetic Predisposition to Disease , Haploinsufficiency , High-Throughput Nucleotide Sequencing , Humans , Kaplan-Meier Estimate , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/mortality , Leukemia, Myeloid, Acute/pathology , Mutation , Myelodysplastic Syndromes/mortality , Myelodysplastic Syndromes/pathology , Myeloproliferative Disorders/mortality , Myeloproliferative Disorders/pathology , Nucleophosmin , Phenotype , Polymorphism, Single Nucleotide , Prognosis , Proportional Hazards Models , Sequence Analysis, DNA/methods , Time Factors
12.
J Clin Invest ; 125(3): 1043-55, 2015 Mar 02.
Article in English | MEDLINE | ID: mdl-25621498

ABSTRACT

BACKGROUND: Mutational inactivation in cancer of key apoptotic pathway components, such as TP53/p53, undermines cytotoxic therapies that aim to increase apoptosis. Accordingly, TP53 mutations are reproducibly associated with poor treatment outcomes. Moreover, cytotoxic treatments destroy normal stem cells with intact p53 systems, a problem especially for myeloid neoplasms, as these cells reverse the low blood counts that cause morbidity and death. Preclinical studies suggest that noncytotoxic concentrations of the DNA methyltransferase 1 (DNMT1) inhibitor decitabine produce p53-independent cell-cycle exits by reversing aberrant epigenetic repression of proliferation-terminating (MYC-antagonizing) differentiation genes in cancer cells. METHODS: In this clinical trial, patients with myelodysplastic syndrome (n=25) received reduced decitabine dosages (0.1-0.2 mg/kg/day compared with the FDA-approved 20-45 mg/m2/day dosage, a 75%-90% reduction) to avoid cytotoxicity. These well-tolerated doses were frequently administered 1-3 days per week, instead of pulse cycled for 3 to 5 days over a 4- to 6-week period, to increase the probability that cancer S-phase entries would coincide with drug exposure, which is required for S-phase-dependent DNMT1 depletion. RESULTS: The median subject age was 73 years (range, 46-85 years), 9 subjects had relapsed disease or were refractory to 5-azacytidine and/or lenalidomide, and 3 had received intensive chemoradiation to treat other cancers. Adverse events were related to neutropenia present at baseline: neutropenic fever (13 of 25 subjects) and septic death (1 of 25 subjects). Blood count improvements meeting the International Working Group criteria for response occurred in 11 of 25 (44%) subjects and were highly durable. Treatment-induced freedom from transfusion lasted a median of 1,025 days (range, 186-1,152 days; 3 ongoing), and 20% of subjects were treated for more than 3 years. Mutations and/or deletions of key apoptosis genes were frequent (present in 55% of responders and in 36% of nonresponders). Noncytotoxic DNMT1 depletion was confirmed by serial BM γ-H2AX (DNA repair/damage marker) and DNMT1 analyses. MYC master oncoprotein levels were markedly decreased. CONCLUSION: Decitabine regimens can be redesigned to minimize cytotoxicity and increase exposure time for DNMT1 depletion, to safely and effectively circumvent mutational apoptotic defects. TRIAL REGISTRATION: Clinicaltrials.gov NCT01165996. FUNDING: NIH (R01CA138858, CA043703); Department of Defense (PR081404); Clinical and Translational Science Award (CTSA) (UL1RR024989); and the Leukemia and Lymphoma Society (Translational Research Program).


Subject(s)
Antimetabolites, Antineoplastic/administration & dosage , Azacitidine/analogs & derivatives , DNA (Cytosine-5-)-Methyltransferases/antagonists & inhibitors , Myelodysplastic Syndromes/drug therapy , Aged , Aged, 80 and over , Azacitidine/administration & dosage , DNA (Cytosine-5-)-Methyltransferase 1 , DNA (Cytosine-5-)-Methyltransferases/metabolism , Decitabine , Drug Administration Schedule , Female , Humans , Male , Middle Aged , Myelodysplastic Syndromes/mortality , Survival Analysis , Treatment Outcome
13.
J Hematol Oncol ; 7: 89, 2014 Dec 07.
Article in English | MEDLINE | ID: mdl-25481243

ABSTRACT

BACKGROUND: The presence of somatic mutations in splicing factor 3b subunit 1 (SF3B1) in patients with Myelodysplastic syndromes with ring sideroblasts (MDS-RS) highlights the importance of the RNA-splicing machinery in MDS. We previously reported the presence of bone marrow (BM) RS in Sf3b1 heterozygous (Sf3b1 (+/-)) mice which are rarely found in mouse models of MDS. Sf3b1 (+/-) mice were originally engineered to study the interaction between polycomb genes and other proteins. METHODS: We used routine blood tests and histopathologic analysis of BM, spleen, and liver to evaluate the hematologic and morphologic characteristics of Sf3b1 (+/-) mice in the context of MDS by comparing the long term follow-up (15 months) of Sf3b1 (+/-) and Sf3b1 (+/+) mice. We then performed a comprehensive RNA-sequencing analysis to evaluate the transcriptome of BM cells from Sf3b1 (+/-) and Sf3b1 (+/+) mice. RESULTS: Sf3b1 (+/-) exhibited macrocytic anemia (MCV: 49.5 ± 1.6 vs 47.2 ± 1.4; Hgb: 5.5 ± 1.7 vs 7.2 ± 1.0) and thrombocytosis (PLTs: 911.4 ± 212.1 vs 878.4 ± 240.9) compared to Sf3b1 (+/+) mice. BM analysis showed dyserythropoiesis and occasional RS in Sf3b1 (+/-) mice. The splenic architecture showed increased megakaryocytes with hyperchromatic nuclei, and evidence of extramedullary hematopoiesis. RNA-sequencing showed higher expression of a gene set containing Jak2 in Sf3b1 (+/-) compared to Sf3b1 (+/+). CONCLUSIONS: Our study indicates that Sf3b1 (+/-) mice manifest features of low risk MDS-RS and may be relevant for preclinical therapeutic studies.


Subject(s)
Anemia, Sideroblastic/genetics , Phosphoproteins/genetics , Ribonucleoprotein, U2 Small Nuclear/genetics , Anemia, Sideroblastic/blood , Animals , Disease Models, Animal , Female , Genotype , Haploinsufficiency/genetics , Humans , Male , Mice , Mice, Transgenic , Phosphoproteins/blood , RNA Splicing , RNA Splicing Factors , Ribonucleoprotein, U2 Small Nuclear/blood , Risk Factors
14.
Exp Biol Med (Maywood) ; 239(7): 853-861, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24879424

ABSTRACT

Cancer chemotherapy is typically toxic. This problem could be addressed by using differences between cancer and normal cells for controlled delivery of drugs to cancer cells. One such difference is the ubiquitously elevated glutathione expression in cancer cells. We report a simple and versatile synthesis of water-soluble gold nanoparticles passivated with amine-containing molecules, which allow for controlled drug release via ligand exchange with bio-available glutathione. Taking methotrexate-passivated gold nanoparticles (Au:MTX) as an example, drug delivery and controlled release via glutathione-mediated ligand exchange was evaluated. Furthermore, the possibility of using Au:MTX to improve therapeutic index in acute myeloid leukemia (AML) models was examined in vitro and in vivo. Au:MTX exhibited cancer selectivity in vitro. Au:MTX had an elevated potency toward an AML cell line THP-1 in a dosage range of 1-5 nM, and therefore an enhanced delivery of drug, whereas normal hematopoietic stem/progenitor cell (HSPC) growth was minimally affected by Au:MTX and MTX treatments within the same range of dosage. In vivo efficacy and safety of Au:MTX was evaluated in a murine xenotransplant model of primary human AML. Au:MTX treatment, compared to control groups including MTX-only and Au nanoparticle-only treatments, produced better leukemia suppression without added toxicity, indicating an enhanced therapeutic index.

15.
J Biol Chem ; 289(21): 14881-95, 2014 May 23.
Article in English | MEDLINE | ID: mdl-24695740

ABSTRACT

Gene activation requires cooperative assembly of multiprotein transcription factor-coregulator complexes. Disruption to cooperative assemblage could underlie repression of tumor suppressor genes in leukemia cells. Mechanisms of cooperation and its disruption were therefore examined for PU.1 and RUNX1, transcription factors that cooperate to activate hematopoietic differentiation genes. PU.1 is highly expressed in leukemia cells, whereas RUNX1 is frequently inactivated by mutation or translocation. Thus, coregulator interactions of Pu.1 were examined by immunoprecipitation coupled with tandem mass spectrometry/Western blot in wild-type and Runx1-deficient hematopoietic cells. In wild-type cells, the NuAT and Baf families of coactivators coimmunoprecipitated with Pu.1. Runx1 deficiency produced a striking switch to Pu.1 interaction with the Dnmt1, Sin3A, Nurd, CoRest, and B-Wich corepressor families. Corepressors of the Polycomb family, which are frequently inactivated by mutation or deletion in myeloid leukemia, did not interact with Pu.1. The most significant gene ontology association of Runx1-Pu.1 co-bound genes was with macrophages, therefore, functional consequences of altered corepressor/coactivator exchange were examined at Mcsfr, a key macrophage differentiation gene. In chromatin immunoprecipitation analyses, high level Pu.1 binding to the Mcsfr promoter was not decreased by Runx1 deficiency. However, the Pu.1-driven shift from histone repression to activation marks at this locus, and terminal macrophage differentiation, were substantially diminished. DNMT1 inhibition, but not Polycomb inhibition, in RUNX1-translocated leukemia cells induced terminal differentiation. Thus, RUNX1 and PU.1 cooperate to exchange corepressors for coactivators, and the specific corepressors recruited to PU.1 as a consequence of RUNX1 deficiency could be rational targets for leukemia differentiation therapy.


Subject(s)
Cell Differentiation/genetics , Co-Repressor Proteins/genetics , Core Binding Factor Alpha 2 Subunit/genetics , Proto-Oncogene Proteins/genetics , Trans-Activators/genetics , Animals , Blotting, Western , Cells, Cultured , Chromatin Immunoprecipitation , Co-Repressor Proteins/metabolism , Core Binding Factor Alpha 2 Subunit/metabolism , Gene Expression Profiling , HEK293 Cells , Humans , Leukemia/genetics , Leukemia/metabolism , Leukemia/pathology , Macrophages/metabolism , Mice , Mice, Knockout , Mutation , Oligonucleotide Array Sequence Analysis , Protein Binding , Proto-Oncogene Proteins/metabolism , Tandem Mass Spectrometry , Trans-Activators/metabolism , Tumor Cells, Cultured
16.
Clin Lymphoma Myeloma Leuk ; 14(2): 172-177.e1, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24332215

ABSTRACT

BACKGROUND: Myelodysplastic syndrome (MDS) continues to cause major morbidity and mortality; thus, novel treatments are needed. The mammalian target of rapamycin (mTOR) inhibitor RAD001 (everolimus) inhibits cellular pathways important to MYC protein stability and cell growth. Pharmacodynamic biomarkers could be useful in distinguishing between (1) disease resistance that occurs even though mTOR is successfully inhibited (suggesting a need for a different treatment strategy) and (2) resistance that might respond to changes in drug dosage or schedule. PATIENTS AND METHODS: This was a small phase II trial of RAD001 in patients with low- and intermediate-1-risk MDS (n = 7). Protein S6K1 (S6) is downstream of mTOR, whereas protein kinase B (AKT) is upstream of mTOR. Therefore, to evaluate the pharmacodynamic effects of RAD001, S6 and AKT phosphorylation (pS6, pAKT) were measured by peripheral blood flow cytometry. RESULTS: Sequential weeks of RAD001 produced a decrease in pS6, whereas pAKT was maintained or increased. There were no clinical responses despite the biomarker evidence of intended pharmacodynamic effect. CONCLUSION: The pS6:pAKT ratio could be useful as a biomarker of target inhibition by RAD001 (clinicaltrials.gov identifier: NCT00809185).


Subject(s)
Biomarkers/metabolism , Myelodysplastic Syndromes/drug therapy , Proto-Oncogene Proteins c-akt/metabolism , Ribosomal Protein S6 Kinases/metabolism , Sirolimus/analogs & derivatives , Aged , Aged, 80 and over , Cell Line, Tumor , Drug Administration Schedule , Dyspnea/chemically induced , Everolimus , Fatigue/chemically induced , Female , Fever/chemically induced , Flow Cytometry , Humans , Immunosuppressive Agents/adverse effects , Immunosuppressive Agents/therapeutic use , Male , Middle Aged , Myelodysplastic Syndromes/metabolism , Neutropenia/chemically induced , Phosphorylation/drug effects , Sirolimus/adverse effects , Sirolimus/pharmacology , Sirolimus/therapeutic use , TOR Serine-Threonine Kinases/metabolism , Treatment Outcome
18.
Clin Cancer Res ; 19(4): 938-48, 2013 Feb 15.
Article in English | MEDLINE | ID: mdl-23287564

ABSTRACT

PURPOSE: The cytidine analogs 5-azacytidine and decitabine, used to treat myelodysplastic syndromes (MDS), produce a molecular epigenetic effect, depletion of DNA-methyltransferase 1 (DNMT1). This action is S-phase dependent. Hence, genetic factors that decrease the half-lives of these drugs could impact efficacy. Documentation of such impact, and elucidation of underlying mechanisms, could lead to improved clinical application. EXPERIMENTAL DESIGN: Cytidine deaminase (CDA) rapidly inactivates 5-azacytidine/decitabine. The effect of CDA SNP A79C and gender on CDA expression, enzyme activity, and drug pharmacokinetics/pharmacodynamics was examined in mice and humans, and the impact on overall survival (OS) was evaluated in 5-azacytidine/decitabine-treated patients with MDS (n = 90) and cytarabine-treated patients with acute myeloid leukemia (AML) (n = 76). RESULTS: By high-performance liquid chromatography (HPLC), plasma CDA activity was decreased as expected in individuals with the SNP A79C. Interestingly and significantly, there was an even larger decrease in females than in males. Explaining this decrease, liver CDA expression was significantly lower in female versus male mice. As expected, decitabine plasma levels, measured by mass spectrometry, were significantly higher in females. In mathematical modeling, the detrimental impact of shorter drug half-life (e.g., in males) was greater in low compared with high S-phase fraction disease (e.g., MDS vs. AML), because in high S-phase fraction disease, even a short exposure treats a major portion of cells. Accordingly, in multivariate analysis, OS was significantly worse in male versus female patients with MDS treated with 5-azacytidine/decitabine. CONCLUSIONS: Increased CDA expression/activity in males contributes to decreased cytidine analog half-life and likely contributes to worse outcomes with 5-azacytidine or decitabine therapy.


Subject(s)
Cytidine Deaminase/genetics , DNA (Cytosine-5-)-Methyltransferases/genetics , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Myelodysplastic Syndromes/drug therapy , Animals , Azacitidine/analogs & derivatives , Cytarabine/administration & dosage , Cytidine/administration & dosage , Cytidine/analogs & derivatives , Cytidine Deaminase/metabolism , DNA (Cytosine-5-)-Methyltransferase 1 , DNA (Cytosine-5-)-Methyltransferases/metabolism , Decitabine , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Leukemia, Myeloid, Acute/pathology , Male , Mice , Myelodysplastic Syndromes/genetics , Myelodysplastic Syndromes/pathology , Polymorphism, Single Nucleotide , Survival Analysis , Treatment Outcome
19.
Oncotarget ; 3(10): 1137-45, 2012 Oct.
Article in English | MEDLINE | ID: mdl-23087155

ABSTRACT

We document for the first time that sanctuary in an organ which expresses high levels of the enzyme cytidine deaminase (CDA) is a mechanism of cancer cell resistance to cytidine analogues. This mechanism could explain why historically, cytidine analogues have not been successful chemotherapeutics against hepatotropic cancers, despite efficacy in vitro. Importantly, this mechanism of resistance can be readily reversed, without increasing toxicity to sensitive organs, by combining a cytidine analogue with an inhibitor of cytidine deaminase (tetrahydrouridine). Specifically, CDA rapidly metabolizes cytidine analogues into inactive uridine counterparts. Hence, to determine if sheltering/protection of cancer cells in organs which express high levels of CDA (e.g., liver) is a mechanism of resistance, we utilized a murine xenotransplant model of myeloid cancer that is sensitive to epigenetic therapeutic effects of the cytidine analogue decitabine in vitro and hepato-tropic in vivo. Treatment of tumor-bearing mice with decitabine (subcutaneous 0.2mg/kg 2X/week) doubled median survival and significantly decreased extra-hepatic tumor burden, but hepatic tumor burden remained substantial, to which the animals eventually succumbed. Combining a clinically-relevant inhibitor of CDA (tetrahydrouridine) with a lower dose of decitabine (subcutaneous 0.1mg/kg 2X/week) markedly decreased liver tumor burden without blood count or bone marrow evidence of myelotoxicity, and with further improvement in survival. In conclusion, sanctuary in a CDA-rich organ is a mechanism by which otherwise susceptible cancer cells can resist the effects of decitabine epigenetic therapy. This protection can be reversed without increasing myelotoxicity by combining tetrahydrouridine with a lower dose of decitabine.


Subject(s)
Antimetabolites, Antineoplastic/therapeutic use , Azacitidine/analogs & derivatives , Cytidine Deaminase/metabolism , Leukemia, Myeloid/enzymology , Liver Neoplasms/enzymology , Liver/enzymology , Animals , Antimetabolites/therapeutic use , Antineoplastic Combined Chemotherapy Protocols , Azacitidine/therapeutic use , Decitabine , Humans , Leukemia, Myeloid/drug therapy , Leukemia, Myeloid/pathology , Liver/drug effects , Liver Neoplasms/drug therapy , Liver Neoplasms/pathology , Mice , Mice, Knockout , Tetrahydrouridine/therapeutic use , Transplantation, Heterologous , Tumor Cells, Cultured , Tumor Suppressor Protein p53/physiology
20.
Syst Biol Reprod Med ; 58(5): 236-44, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22632096

ABSTRACT

We explored the relationship between sperm chromatin integrity, hormone levels, seminal plasma total antioxidant capacity (TAC), and routine sperm parameters in men with male factor (MF, n = 81) and non-male factor (NMF, n = 52) infertility. Semen and blood were collected and examined from men undergoing evaluation for infertility in the Avicenna Infertility Clinic. We have examined each patient for serum hormones (LH, FSH, E2, DHEA), sperm chromatin damage, level of protamination and seminal plasma TAC. Levels of FSH, LH, sperm chromatin damage, and abnormal protamination were significantly higher in MF vs. NMF groups (p < 0.001). Sperm chromatin damage was correlated with percentage of CMA(3)- positive sperm (r = 0.64, p < 0.001) and with sperm concentration (r = -0.36, p < 0.001), motility (r = -0.21, p < 0.05), and morphologically normal spermatozoa (r = -0.29, p < 0.001). Linear regression showed sperm chromatin damage was related to percentage of CMA(3)- positive sperm (p < 0.001) in ungrouped patients. It was related to both percentage of CMA(3)- positive sperm and serum DHEA in the MF group (p < 0.001 and p < 0.05, respectively). Sperm chromatin maturity assessed by CMA(3) test was inversely related to sperm chromatin damage assessed by the toludine blue assay. Male factor infertility associated with sperm chromatin damage may be related to sperm protamination and to serum DHEA.


Subject(s)
Antioxidants/analysis , Chromatin Assembly and Disassembly , Hormones/blood , Infertility, Male/diagnosis , Semen/chemistry , Spermatozoa/metabolism , Adolescent , Adult , Biomarkers/blood , Dehydroepiandrosterone/blood , Estradiol/blood , Follicle Stimulating Hormone, Human/blood , Humans , Infertility, Male/blood , Infertility, Male/genetics , Infertility, Male/pathology , Iran , Linear Models , Luteinizing Hormone/blood , Male , Middle Aged , Multivariate Analysis , Semen Analysis , Spermatozoa/pathology , Testosterone/blood , Young Adult
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